Modified Project Summary/Abstract Plexins are transmembrane receptors that make cell guidance decisions in vascular and neuronal development, disease, and regeneration. Targeting plexins and their co-receptors, especially neuropilin, is a novel route to treating retinal diseases such as age-related macular degeneration (AMD) and diabetic retinopathy, but the mechanistic details of plexin activation are poorly understood. We will characterize the interactions between plexin and membrane lipids. Plexins are unique transmembrane receptors in that their intracellular regions interact directly with membrane-anchored small GTPases. These Ras and Rhofamily GTPases, in turn, are associated with cell migration in the retina. The preliminary results in aim 1 indicate that protein-membrane interactions play a key role in the signaling mechanisms of plexins, for example, rationalizing the different functional roles of the GTPases in the system. Our work will be the first to study the interactions of plexin domains with the lipid bilayer at the molecular level. This includes, in aim1, the plexin-B1 transmembrane region which has been predicted to assume three states of inter helix contacts. Altogether the results support a multi-state model which incorporates previous and the new results, but also points to critical new interactions between the proteins and the lipid bilayer. Aim 2 seeks to resolve the structure and function of the transmembrane regions of plexin coreceptors, neuropilin and semaphorins. We hypothesize that such interactions set up cross signaling with ligands and co-receptors. In order to address this hypothesis, we will examine receptor homo- and hetero-oligomerization using time resolved fluorescence spectroscopy and functional experiments, both using live cells. Together with NMR and molecular dynamics, these complementary tools will address the hypothesis at different levels of complexity, ranging from domain level to in-cell behavior. Plexin function is modulated by interaction with co-receptors, such as neuropilins which are also single pass transmembrane (TM) proteins. TM regions are recently recognized as an effective target for peptides designed to disrupt receptor signaling. In aim 2, we will characterize the structure and function of plexin TM interactions with the TM domains of neuropilins. Our data suggest that TM interactions between plexins and certain of their semaphorin ligands can also occur in cis. Such TM based regulation would be a novel mechanism in the plexin field, and one that can be targeted to inhibit the receptors in the retina. Specifically, expanding the successful use of neuropilin peptides as anti-tumor agents, TM regions are a target for anti-angiogenesis therapy in eye disease such as AMD and diabetic retinopathy.